Subpicomolar diphenyleneiodonium inhibits microglial NADPH oxidase with high specificity and shows great potential as a therapeutic agent for neurodegenerative diseases

Abstract

Activation of microglial NADPH oxidase (NOX2) plays a critical role in mediating neuroinflammation, which is closely linked with the pathogenesis of a variety of neurodegenerative diseases, including Parkinson's disease (PD). The inhibition of NOX2-generated superoxide has become an effective strategy for developing disease-modifying therapies for PD. However, the lack of specific and potent NOX2 inhibitors has hampered the progress of this approach. Diphenyleneiodonium (DPI) is a widely used, long-acting NOX2 inhibitor. However, due to its non-specificity for NOX2 and high cytotoxicity at standard doses (µM), DPI has been precluded from human studies. In this study, using ultra-low doses of DPI, we aimed to: (1) investigate whether these problems could be circumvented and (2) determine whether ultra-low doses of DPI were able to preserve its utility as a potent NOX2 inhibitor. We found that DPI at subpicomolar concentrations (10(-14) and 10(-13) M) displays no toxicity in primary midbrain neuron-glia cultures. More importantly, we observed that subpicomolar DPI inhibited phorbol myristate acetate (PMA)-induced activation of NOX2. The same concentrations of DPI did not inhibit the activities of a series of flavoprotein-containing enzymes. Furthermore, potent neuroprotective efficacy was demonstrated in a post-treatment study. When subpicomolar DPI was added to neuron-glia cultures pretreated with lipopolysaccharide, 1-methyl-4-phenylpyridinium or rotenone, it potently protected the dopaminergic neurons. In summary, DPI's unique combination of high specificity toward NOX2, low cytotoxicity and potent neuroprotective efficacy in post-treatment regimens suggests that subpicomolar DPI may be an ideal candidate for further animal studies and potential clinical trials.

Keywords: NADPH oxidase; Parkinson's disease; microglia; neuroinflammation; oxidative stress.

Figure 1

Effects of DPI on cell viability and DA uptake capacity in primary midbrain neuron-glia cultures incubated with different concentrations of DPI for 48 h. (A) Cell viability was evaluated by MTT assays. (B) Representative images of cells immunostained with Neu-N, Iba-1 and GFAP antibodies indicate lesions on the neurons, microglia and astroglia after micromolar, but not subpicomolar, DPI exposure. The inserts show amplified microglia in each group. (C) [³H]-DA uptake analysis revealed a decrease in neurotransmitter uptake capacity after micromolar, but not subpicomolar, DPI exposure. The results are expressed as a percentage of the controls (mean ± SEM) from three experiments performed in duplicate and were analyzed using one-way ANOVA, followed by Bonferroni’s post hoc multiple comparison test. **p < 0 .01; Bar = 50 μm.

Figure 2

Subpicomolar DPI displays specificity for NOX2. (A–D) DPI at 10⁻¹³ and 10⁻¹⁴ M fails to inhibit commercially purified (A) iNOS (nitrite production as an index), (B) xanthine oxidase, (C) cytochrome P450 reductase and (D) thioredoxin reductase, although DPI at micromolar concentrations decreases these enzyme activities. Data are expressed as the mean ± SEM from three to four experiments performed in duplicate. (E–J) The effects of DPI on the enzymatic activities of NOX2, iNOS, xanthine oxidase, cytochrome P450 reductase, thioredoxin reductase and NADH-ubiquinone oxidoreductase in neuron-glia cultures. (E) Cellular NOX2 activation was induced by PMA in neuron-glia cultures. Superoxide production was used as an index of NOX2 activity. The addition of 10⁻¹³ or 10⁻¹⁴ M DPI inhibits NOX2-generated superoxide as efficiently as micromolar concentrations, indicating NOX2 inhibition. Data are expressed as a percentage of the PMA group (mean ± SEM) from three to four experiments performed in duplicate. (F) Cellular iNOS was induced in neuron-glia cultures by incubation with LPS for 12 h. Unlike micromolar concentrations, DPI at 10⁻¹³ and 10⁻¹⁴ M fails to reduce the generation of iNOS-generated nitrite. (G–J) DPI at 10⁻⁵ M, but not 10⁻¹³ and 10⁻¹⁴ M, inhibits xanthine oxidase, cytochrome P450 reductase, thioredoxin reductase and NADH-ubiquinone oxidoreductase in neuron-glia cultures. Data are expressed as the mean ± SEM from three to four experiments performed in duplicate. The results were analyzed using one-way ANOVA, followed by Bonferroni’s post hoc multiple comparison test. **p < 0.01. XO, xanthine oxidase; CPR, cytochrome P450 reductase; TR, thioredoxin reductase (TR); NUO, NADH-ubiquinone oxidoreductase.

Figure 3

Dopaminergic neuroprotection by post-treatment with subpicomolar DPI 12 h after inflammatory challenge in primary neuron-glia cultures. (A) Experimental designs. Midbrain neuron-glia cultures were pre-treated with LPS (20 ng/ml) for 12 h, followed by DPI (10⁻¹⁴ or 10^{− 13} M) treatment. (B) [³H]-DA uptake assay and (C) THir neuron count analysis revealed significant dopaminergic protection 7 days after DPI treatment. (D) Representative cell images of TH immunostaining 7 days after DPI treatment indicate prominent protection of the dopaminergic neuronal cell bodies and dendrites. The results of DA uptake are expressed as a percentage of the controls and are the mean ± SEM from three to four experiments performed in duplicate. The results of THir cell counts are expressed as the mean ± SEM from three to four experiments performed in duplicate. Data were analyzed using one-way ANOVA, followed by Bonferroni’s post hoc multiple comparison test. **p < 0.01; Bar = 50 μm.

Figure 4

Dopaminergic neuroprotection by post-treatment with subpicomolar DPI 24 h after xenobiotic damage in primary neuron-glia cultures. (A) Experimental designs. Midbrain neuron-glia cultures were pre-treated with LPS (20 ng/ml), MPP⁺ (0.15 μM) or rotenone (10 nM) for 24 h, followed by DPI (10⁻¹⁴ or 10⁻¹³ M) treatment. (B–D) Seven days after DPI treatment, significant dopaminergic protection was observed using the [³H]-DA uptake assay. The results are expressed as a percentage of the controls and are the mean ± SEM from three to four experiments performed in duplicate. Data were analyzed using one-way ANOVA, followed by Bonferroni’s post hoc multiple comparison test. *p < 0.05, **p < 0.01.

Figure 5

Post-treatment with subpicomolar DPI attenuates NOX2 activation induced by LPS through detachment of p47^phox from the plasma membrane. (A) Midbrain neuron-glia cultures were pre-treated with LPS for 12 h, followed by DPI (10⁻¹⁴ or 10⁻¹³ M) treatment. Superoxide production was significantly inhibited by subpicomolar DPI post-treatment. (B) Western blot analysis revealed that subpicomolar DPI post-treatment detaches p47^phox from the plasma membrane in LPS-treated HAPI microglia cells (gp91^phox as an internal membrane control). The densities of the membrane p47^phox signals were quantified. The results are expressed as a percentage of the LPS group (mean ± SEM) from three to four experiments performed in duplicate and were analyzed using one-way ANOVA, followed by Bonferroni’s post hoc multiple comparison test. **p < 0.01.